In the past few years, microorganisms have proved to be capable of producing foreign peptides and proteins, encoded by foreign genes artificially introduced by means of a transformation system and expressed under the control of regulatory sequences.
Some of the basic techniques for this procedure have been disclosed in, for example, U.S. Pat. No. 4,237,224.
The basic constituents of recombinant DNA technology are formed by:
the gene encoding the desired property and provided with adequate control sequences required for expression in the host organism,
a vector, usually a plasmid, into which the gene can be inserted to guarantee stable replication and a high level of expression of the said gene,
a suitable host microorganism into which the vector carrying the said gene can be transformed and having the cellular systems to express the information of the said gene.
Amongst the products thus formed are enzymes, hormones, antigens and other useful peptides and proteins.
Some of these products are used as pharmaceutical agents, e.g. growth hormone and interferon, others as auxiliaries in the food industry e.g. beta-galactosidase (lactase), chymosin and amyloglucosidase, and still others may act as biological catalysts for the conversion of certain compounds.
Every contamination of pharmaceuticals or food with harmful organisms or substances should be excluded. The host organisms should also be harmless to persons handling the microbes during experimentation or large scale production processes. Therefore, a prerequisite for the host is that it is not pathogenic.
The first years of recombinant-DNA work were characterized by stringent rules and restrictions to prevent or limit undesired side effects, especially the uncontrolled dissemination of pathogenic microorganisms in the environment.
Although the concern about the supposed risks seems to have been exaggerated, there still remains a steady need for hosts which are not associated with any noxious effect.
Up to now, commercial efforts involving recombinant genetic manipulation of plasmids for producing various substances have centered on Escherichia coli as a host organism. The main reason is that E. coli is historically the best studied microorganism. The first discoveries and inventions made in recombinant DNA technology have been made with E. coli as the host.
However, E. coli is not the most desirable organism to use for commercial production of substances applied in pharmaceutical and food industry. It may even prove to be unsuitable as a host/vector system in some situations, because of the presence of a number of toxic pyrogenic factors. The elimination of these may often cause problems. Therefore, E. coli has only a very limited use as production organism in fermentation industry. Also the proteolytic activities in E. coli may seriously limit yields of some useful products.
These and other considerations have led to an increased interest in alternative host/vector systems. The interest is concentrating in particular on the use of eukaryotic systems for the production of eukaryotic products. A continuing demand exists for new hosts which are above any suspicion as production organisms for chemical substances, in particular food-grade and pharmaceutical grade products, and which moreover are suited to large scale fermentations in industry.
The names of many harmless microorganisms are found on the so called GRAS (Generally Recognized as Safe) list. However, only few genetic procedures are known sofar for the cloning and expression of genes in GRAS-organisms.
Amongst the eukaryotic organisms suitable for commercial exploitation yeasts are perhaps the easiest ones to manage. Yeast, especially bakers' yeast, is relatively cheap, easy to grow in large quantities and has a highly developed genetic system.
The term yeast is frequently used to indicate only Saccharomyces cerevisiae or bakers' yeast, which is one of the most common and well-known species. It will be understood that the term yeast as used in this specification is meant to indicate all genera and is not restricted to the species Saccharomyces cerevisiae.
Recently, it has been disclosed that cells of Saccharomyces cerevisiae are susceptible to transformation by plasmids (A. Hinnen et al., Proc. Natl. Acad. Sci. USA 75 (1978), 1929). Success has been had with cloning and expressing in this yeast the bacterial resistance genes for ampicillin, chloramphenicol and kanamycin, but also eukaryotic genes like the lactase gene and the heterologous genes for ovalbumin, leukocyte interferon D and also a Drosophila gene (see review C. P. Hollenberg, Current Topics in microbiology and Immunology, 96 (1982) 119-144).
Up to now, only one other yeast species has been investigated as a host for yeast expression vectors. The Saccharomyces cerevisiae leucine gene has been successfully cloned and expressed in Schizosaccharomyces pombe (D. Beach, and P. Nurse, Nature 290 (1981) 140-142).
Yeast vectors which have been described to give successful transformation are based on the natural 2 .mu.m (2 micron) plasmid occurring in many strains of S. cerevisiae (see e.g. J. D. Beggs, Nature 275 (1978), 104-109), and on the autonomously replicating sequences (ARS) derived from yeast chromosomal DNA (see e.g. K. Struhl et al., Proc. Natl. Acad. Sci. USA 76 (1979), 1035-1039), respectively. Vectors for Saccharomyces cerevisiae which can be used for transformation purposes have also been described by C. P. Hollenberg, Current Topics in Microbiology and Immunology, 96 (1982) 119-144.
The transformation of not well characterized or industrial yeast species is severely hampered by the lack of knowledge about transformation conditions and suitable vectors. In addition, auxotrophic markers are often not available or are undesired, precluding a direct selection by auxotrophic complementation.